title: The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation.
abstract: Full genome sequencing of bacterial genomes has revealed the presence of numerous genes encoding family X DNA polymerases. These enzymes play a variety of biological roles and, accordingly, display often striking functional differences. Here we report that the PolX from the heat-stable organism Thermus thermophilus (TthPolX) inserts the four dNTPs with strong asymmetry. We demonstrate that this behaviour is related to the presence of a single divergent residue in the active site of TthPolX. Mutation of this residue (Ser(266)) to asparagine, the residue present in most PolXs, had a strong effect on TthPolX polymerase activity, increasing and equilibrating the insertion efficiencies of the 4 dNTPs. Moreover, we show that this behaviour correlates with the ability of TthPolX to insert 8-oxo-dGMP. Although the wild-type enzyme inefficiently incorporates 8-oxo-dGMP, the substitution of Ser(266) to asparagine resulted in a dramatic increase in 8-oxo-dGMP incorporation opposite dA. These results suggest that the presence of a serine at position 266 in TthPolX allows the enzyme to minimize the formation of dA:8-oxo-dGMP at the expense of decreasing the insertion rate of pyrimidines. We discuss the structural basis for these effects and the implications of this behaviour for the GO system (BER of 8-oxo-dG lesions).
authors.affiliation: X-Pol Biotech S.L.U. Parque Científico de Madrid. Cantoblanco, Madrid 28049, Spain, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA and Centro de Biología Molecular Severo Ochoa (CSIC-UAM). Cantoblanco, Madrid 28049, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Garrido
authors.forename: Edison
authors.initials: E
authors.lastname: Mejia
authors.forename: Miguel
authors.initials: M
authors.lastname: Garcia-Diaz
authors.forename: Luis
authors.initials: L
authors.lastname: Blanco
authors.forename: Angel J
authors.initials: AJ
authors.lastname: Picher
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; C078206:8-oxodeoxyguanosine triphosphate; D012694:Serine; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase